Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566616
Tadej Markuš, Mladen Soldat, Vasilka Magdevska, Jaka Horvat, Martin Kavšček, Gregor Kosec, Štefan Fujs, Uroš Petrovič
Yarrowia lipolytica is an oleaginous yeast with ever growing popularity in the metabolic engineering circles. It is well known for its ability to accommodate a high carbon flux through acetyl-CoA and is being extensively studied for production of chemicals derived from it. We investigated the effects of modifying the upstream metabolism leading to acetyl-CoA on beta-carotene production, including its titer, yield, and content. We examined the pyruvate and the phosphoketolase bypass, both of which are stoichiometrically favorable for the production of acetyl-CoA and beta-carotene. Additionally, we examined a set of genes involved in the carnitine shuttle. We constructed a set of parental strains derived from the Y. lipolytica YB-392 wild-type strain, each with a different capacity for beta-carotene production, and introduced genes for the metabolic bypasses in each of the constructed parental strains. Subsequently, we subjected these constructed strains to a series of fermentation experiments. We discovered that altering the upstream metabolism in most cases led to a decrease in performance for production of beta-carotene. Most notably, a set of genes used for the pyruvate bypass ( YlPDC2 , YlALD5 , and YlACS1 ) and the phosphoketolase bypass ( LmXPK and CkPTA ) resulted in the reduction of more than 30%. Our findings contribute to our understanding of Y. lipolytica 's metabolic capacity and suggest that production of beta-carotene is most likely not limited solely by the acetyl-CoA supply. We also highlight a complex nature of engineering Y. lipolytica , as most of the results from studies using a different strain background did not align with our findings.
{"title":"Impact of central carbon metabolism bypasses on the production of beta-carotene in<i>Yarrowa lipolytica</i>","authors":"Tadej Markuš, Mladen Soldat, Vasilka Magdevska, Jaka Horvat, Martin Kavšček, Gregor Kosec, Štefan Fujs, Uroš Petrovič","doi":"10.1101/2023.11.10.566616","DOIUrl":"https://doi.org/10.1101/2023.11.10.566616","url":null,"abstract":"Yarrowia lipolytica is an oleaginous yeast with ever growing popularity in the metabolic engineering circles. It is well known for its ability to accommodate a high carbon flux through acetyl-CoA and is being extensively studied for production of chemicals derived from it. We investigated the effects of modifying the upstream metabolism leading to acetyl-CoA on beta-carotene production, including its titer, yield, and content. We examined the pyruvate and the phosphoketolase bypass, both of which are stoichiometrically favorable for the production of acetyl-CoA and beta-carotene. Additionally, we examined a set of genes involved in the carnitine shuttle. We constructed a set of parental strains derived from the Y. lipolytica YB-392 wild-type strain, each with a different capacity for beta-carotene production, and introduced genes for the metabolic bypasses in each of the constructed parental strains. Subsequently, we subjected these constructed strains to a series of fermentation experiments. We discovered that altering the upstream metabolism in most cases led to a decrease in performance for production of beta-carotene. Most notably, a set of genes used for the pyruvate bypass ( YlPDC2 , YlALD5 , and YlACS1 ) and the phosphoketolase bypass ( LmXPK and CkPTA ) resulted in the reduction of more than 30%. Our findings contribute to our understanding of Y. lipolytica 's metabolic capacity and suggest that production of beta-carotene is most likely not limited solely by the acetyl-CoA supply. We also highlight a complex nature of engineering Y. lipolytica , as most of the results from studies using a different strain background did not align with our findings.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"37 12","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992980","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.566975
Carl L McCombe, Alex Wegner, Chenie S. Zamora, Florencia Casanova, Shouvik Aditya, Julian R Greenwood, Louisa Wirtz, Samuel de Paula, Eleanor England, Sascha Shang, Daniel J Ericsson, Ely Oliveira-Garcia, Simon J Williams, Ulrich Schaffrath
Phosphate availability modulates plant immune function and regulates interactions with beneficial, phosphate-providing, microbes. Here, we describe the hijacking of plant phosphate sensing by a family of Nudix hydrolase effectors from pathogenic Magnaporthe oryzae and Colletotrichum fungi. Structural and enzymatic analyses of the Nudix effector family demonstrate that they selectively hydrolyze inositol pyrophosphates, a molecule used by plants to monitor phosphate status and regulate starvation responses. In M. oryzae , gene deletion and complementation experiments reveal that the enzymatic activity of a Nudix effector significantly contributes to pathogen virulence. Further, we show that this conserved effector protein family induces phosphate starvation signaling in plants. Our study elucidates a molecular mechanism, utilized by multiple phytopathogenic fungi, that manipulates the highly conserved plant phosphate sensing pathway to exacerbate disease.
{"title":"Plant pathogenic fungi hijack phosphate starvation signaling with conserved enzymatic effectors","authors":"Carl L McCombe, Alex Wegner, Chenie S. Zamora, Florencia Casanova, Shouvik Aditya, Julian R Greenwood, Louisa Wirtz, Samuel de Paula, Eleanor England, Sascha Shang, Daniel J Ericsson, Ely Oliveira-Garcia, Simon J Williams, Ulrich Schaffrath","doi":"10.1101/2023.11.14.566975","DOIUrl":"https://doi.org/10.1101/2023.11.14.566975","url":null,"abstract":"Phosphate availability modulates plant immune function and regulates interactions with beneficial, phosphate-providing, microbes. Here, we describe the hijacking of plant phosphate sensing by a family of Nudix hydrolase effectors from pathogenic Magnaporthe oryzae and Colletotrichum fungi. Structural and enzymatic analyses of the Nudix effector family demonstrate that they selectively hydrolyze inositol pyrophosphates, a molecule used by plants to monitor phosphate status and regulate starvation responses. In M. oryzae , gene deletion and complementation experiments reveal that the enzymatic activity of a Nudix effector significantly contributes to pathogen virulence. Further, we show that this conserved effector protein family induces phosphate starvation signaling in plants. Our study elucidates a molecular mechanism, utilized by multiple phytopathogenic fungi, that manipulates the highly conserved plant phosphate sensing pathway to exacerbate disease.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"36 11","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566588
Alexander Sasse, Maria Chikina, Sara Mostafavi
To understand the decision process of genomic sequence-to-function models, various explainable AI algorithms have been proposed. These methods determine the importance of each nucleotide in a given input sequence to the model's predictions, and enable discovery of cis regulatory motif grammar for gene regulation. The most commonly applied method is in silico saturation mutagenesis (ISM) because its per-nucleotide importance scores can be intuitively understood as the computational counterpart to in vivo saturation mutagenesis experiments. While ISM is highly interpretable, it is computationally challenging to perform, because it requires computing three forward passes for every nucleotide in the given input sequence; these computations add up when analyzing a large number of sequences, and become prohibitive as the length of the input sequences and size of the model grows. Here, we show how to use the first-order Taylor approximation for ISM, which reduces its computation cost to a single forward pass for an input sequence, placing its scalability on equal footing with gradient-based approximation methods such as "gradient-times-input". We show that the Taylor ISM (TISM) approximation is robust across different model ablations, random initializations, training parameters, and data set sizes. We use our theoretical derivation to connect ISM with the gradient values and show how this approximation is related to a recently suggested correction of the model's gradients.
为了理解基因组序列到功能模型的决策过程，人们提出了各种可解释的人工智能算法。这些方法确定了给定输入序列中每个核苷酸对模型预测的重要性，并能够发现基因调控的顺式调控基序语法。最常用的方法是硅饱和诱变(ISM)，因为它的每核苷酸重要性评分可以直观地理解为体内饱和诱变实验的计算对应。虽然ISM是高度可解释性的，但它在计算上很难执行，因为它需要为给定输入序列中的每个核苷酸计算三次正向传递;当分析大量序列时，这些计算加起来，并且随着输入序列的长度和模型大小的增长而变得令人望而却步。在这里，我们展示了如何使用ISM的一阶泰勒近似，它将输入序列的计算成本降低到单个前向传递，将其可扩展性与基于梯度的近似方法(如“梯度-时间-输入”)置于同等地位。我们证明了Taylor ISM (TISM)近似在不同的模型衰减、随机初始化、训练参数和数据集大小上都是鲁棒的。我们使用我们的理论推导将ISM与梯度值联系起来，并展示了这种近似是如何与最近建议的模型梯度修正相关联的。
{"title":"Quick and effective approximation of in silico saturation mutagenesis experiments with first-order Taylor expansion","authors":"Alexander Sasse, Maria Chikina, Sara Mostafavi","doi":"10.1101/2023.11.10.566588","DOIUrl":"https://doi.org/10.1101/2023.11.10.566588","url":null,"abstract":"To understand the decision process of genomic sequence-to-function models, various explainable AI algorithms have been proposed. These methods determine the importance of each nucleotide in a given input sequence to the model's predictions, and enable discovery of cis regulatory motif grammar for gene regulation. The most commonly applied method is in silico saturation mutagenesis (ISM) because its per-nucleotide importance scores can be intuitively understood as the computational counterpart to in vivo saturation mutagenesis experiments. While ISM is highly interpretable, it is computationally challenging to perform, because it requires computing three forward passes for every nucleotide in the given input sequence; these computations add up when analyzing a large number of sequences, and become prohibitive as the length of the input sequences and size of the model grows. Here, we show how to use the first-order Taylor approximation for ISM, which reduces its computation cost to a single forward pass for an input sequence, placing its scalability on equal footing with gradient-based approximation methods such as \"gradient-times-input\". We show that the Taylor ISM (TISM) approximation is robust across different model ablations, random initializations, training parameters, and data set sizes. We use our theoretical derivation to connect ISM with the gradient values and show how this approximation is related to a recently suggested correction of the model's gradients.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"36 10","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992992","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566787
Alexandru M Plesa, Sascha Jung, Helen H Wang, Fawad Omar, Michael Shadpour, David Choy Buentello, Maria C Perez-Matos, Naftali Horwitz, George Cai, Zhen-Kai Ngian, Carol V de Magalhaes, Amy J Wagers, William B Mair, Antonio del Sol Mesa, George M Church
Aging is a complex process that manifests through the time-dependent functional decline of a biological system. Age-related changes in epigenetic and transcriptomic profiles have been successfully used to measure the aging process 1,2 . Moreover, modulating gene regulatory networks through interventions such as the induction of the Yamanaka factors has been shown to reverse aging signatures and improve cell function 3,4 . However, this intervention has safety and efficacy limitations for in vivo rejuvenation 5,6 , underscoring the need for identifying novel age reversal factors. Here, we discovered SRSF1 as a new rejuvenation factor that can improve cellular function in vitro and in vivo . Using a cDNA overexpression screen with a transcriptomic readout we identified that SRSF1 induction reprograms the cell transcriptome towards a younger state. Furthermore, we observed beneficial changes in senescence, proteasome function, collagen production, and ROS stress upon SRSF1 overexpression. Lastly, we showed that SRSF1 can improve wound healing in vitro and in vivo and is linked to organismal longevity. Our study provides a proof of concept for using transcriptomic reprogramming screens in the discovery of age reversal interventions and identifies SRSF1 as a promising target for cellular rejuvenation.
{"title":"Transcriptomic reprogramming screen identifies SRSF1 as rejuvenation factor","authors":"Alexandru M Plesa, Sascha Jung, Helen H Wang, Fawad Omar, Michael Shadpour, David Choy Buentello, Maria C Perez-Matos, Naftali Horwitz, George Cai, Zhen-Kai Ngian, Carol V de Magalhaes, Amy J Wagers, William B Mair, Antonio del Sol Mesa, George M Church","doi":"10.1101/2023.11.13.566787","DOIUrl":"https://doi.org/10.1101/2023.11.13.566787","url":null,"abstract":"Aging is a complex process that manifests through the time-dependent functional decline of a biological system. Age-related changes in epigenetic and transcriptomic profiles have been successfully used to measure the aging process 1,2 . Moreover, modulating gene regulatory networks through interventions such as the induction of the Yamanaka factors has been shown to reverse aging signatures and improve cell function 3,4 . However, this intervention has safety and efficacy limitations for in vivo rejuvenation 5,6 , underscoring the need for identifying novel age reversal factors. Here, we discovered SRSF1 as a new rejuvenation factor that can improve cellular function in vitro and in vivo . Using a cDNA overexpression screen with a transcriptomic readout we identified that SRSF1 induction reprograms the cell transcriptome towards a younger state. Furthermore, we observed beneficial changes in senescence, proteasome function, collagen production, and ROS stress upon SRSF1 overexpression. Lastly, we showed that SRSF1 can improve wound healing in vitro and in vivo and is linked to organismal longevity. Our study provides a proof of concept for using transcriptomic reprogramming screens in the discovery of age reversal interventions and identifies SRSF1 as a promising target for cellular rejuvenation.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"45 21","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134902666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.566982
Ines Maria Alonso-Crespo, Vicky M. Temperton, Andreas Fichtner, Thomas Niemeyer, Michael Schloter, Benjamin M. Delory
1) The order of arrival of plant species during community assembly can affect how species interact with each other. These so-called priority effects can have strong implications for the structure and functioning of plant communities. However, the extent to which the strength, direction, and persistence of priority effects are modulated by weather conditions during plant establishment ("year effects") is not well known. 2) Here we present the first results from a long-term field experiment (POEM: PriOrity Effects Mechanisms) initiated in 2020 in Northern Germany to test how plant functional group (PFG) order of arrival and the year of initiation of an experiment interactively affect the structure and functioning of nutrient-poor dry acidic grasslands, both above and belowground. To do this, we established the same experiment, manipulating the order of arrival of forbs, grasses and legumes on the same site, but in different years. 3) We found that time since establishment was a stronger driver of plant community composition than PFG order of arrival and year of initiation. These three factors interactively affected plant species diversity, with the effect of PFG order of arrival on plant species richness depending on time since establishment. Year of initiation, not PFG order of arrival, was the strongest driver of aboveground community productivity. Although we did not find any effect of PFG order of arrival on root productivity, it had a strong impact on the vertical distribution of roots. Communities where grasses were sown first rooted more shallowly than communities in which forbs or legumes were sown first. 4) Synthesis: Our results demonstrate that plant order of arrival and year effects jointly affect plant diversity and species composition, with time since establishment also playing an important role. While year effects were more important than plant order of arrival in modulating aboveground biomass production in our nutrient-poor grassland, we showed that plant order of arrival can strongly affect the vertical distribution of roots, with communities in which forbs or legumes were sown first rooting deeper than grasses-first communities. These results suggest that a deeper understanding of priority and year effects is needed to better predict restoration outcomes.
{"title":"Exploring priority and year effects on plant diversity, productivity and vertical root distribution: first insights from a grassland field experiment","authors":"Ines Maria Alonso-Crespo, Vicky M. Temperton, Andreas Fichtner, Thomas Niemeyer, Michael Schloter, Benjamin M. Delory","doi":"10.1101/2023.11.14.566982","DOIUrl":"https://doi.org/10.1101/2023.11.14.566982","url":null,"abstract":"1) The order of arrival of plant species during community assembly can affect how species interact with each other. These so-called priority effects can have strong implications for the structure and functioning of plant communities. However, the extent to which the strength, direction, and persistence of priority effects are modulated by weather conditions during plant establishment (\"year effects\") is not well known. 2) Here we present the first results from a long-term field experiment (POEM: PriOrity Effects Mechanisms) initiated in 2020 in Northern Germany to test how plant functional group (PFG) order of arrival and the year of initiation of an experiment interactively affect the structure and functioning of nutrient-poor dry acidic grasslands, both above and belowground. To do this, we established the same experiment, manipulating the order of arrival of forbs, grasses and legumes on the same site, but in different years. 3) We found that time since establishment was a stronger driver of plant community composition than PFG order of arrival and year of initiation. These three factors interactively affected plant species diversity, with the effect of PFG order of arrival on plant species richness depending on time since establishment. Year of initiation, not PFG order of arrival, was the strongest driver of aboveground community productivity. Although we did not find any effect of PFG order of arrival on root productivity, it had a strong impact on the vertical distribution of roots. Communities where grasses were sown first rooted more shallowly than communities in which forbs or legumes were sown first. 4) Synthesis: Our results demonstrate that plant order of arrival and year effects jointly affect plant diversity and species composition, with time since establishment also playing an important role. While year effects were more important than plant order of arrival in modulating aboveground biomass production in our nutrient-poor grassland, we showed that plant order of arrival can strongly affect the vertical distribution of roots, with communities in which forbs or legumes were sown first rooting deeper than grasses-first communities. These results suggest that a deeper understanding of priority and year effects is needed to better predict restoration outcomes.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"40 13","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992521","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.564735
Lauriane Michel, Hrag Esfahani, Roxane Verdoy, Delphine de Mulder, Jerome Ambroise, Veronique Roelants, Bertrand Bouchard, Jerome Savary, Joseph Dewulf, Thomas Doumont, Caroline Bouzin, Vincent Haufroid, Joost J.F.P. Luiken, Miranda Nabben, Michael Singleton, Luc Bertrand, Matthieu Ruiz, Christine Des Rosiers, Jean-Luc Balligand
Background: Cardiac beta3-adrenergic receptors (beta3AR) are upregulated in diseased hearts and mediate antithetic effects to those of beta1AR and beta2AR. Beta3AR agonists were recently shown to protect from myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. The underlying mechanisms, however, remain elusive. Methods: To dissect functional, transcriptional and metabolic effects, hearts and isolated ventricular myocytes from mice harboring a moderate, cardiac-specific expression of a human ADRB3 transgene (beta3AR-Tg) and subjected to transverse aortic constriction (TAC) were assessed using echocardiography, RNAseq, PET scan, metabolomics, seahorse and metabolic flux analysis. Subsequently, signaling and metabolic pathways were investigated further in vivo in beta3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express beta3AR and subjected to neurohormonal stress. These results were completed with an analysis of single nucleus RNAseq data from human cardiac myocytes from heart failure patients. Results: Compared with WT littermate, beta3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. Beta3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the Pentose-Phosphate Pathway (PPP) in beta3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the PPP, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, while downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in beta3AR-Tg post-TAC compared to WT, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as upstream transcription factor which was functionally verified in beta3AR- expressing cardiac myocytes where its translocation and nuclear activity was dependent on beta3AR activation of nitric-oxide synthase (NOS) NO production. Conclusion: Moderate expression of cardiac beta3AR, at levels observed in human cardiac myocardium, exerts antioxidant effects through activation of the PPP and NRF2 pathway, thereby preserving myocardial oxidative metabolism, function and integrity under pathophysiological stress.
{"title":"A NRF2/beta3-adrenoreceptor axis drives a sustained antioxidant and metabolic rewiring through the pentose-phosphate pathway to alleviate cardiac stress","authors":"Lauriane Michel, Hrag Esfahani, Roxane Verdoy, Delphine de Mulder, Jerome Ambroise, Veronique Roelants, Bertrand Bouchard, Jerome Savary, Joseph Dewulf, Thomas Doumont, Caroline Bouzin, Vincent Haufroid, Joost J.F.P. Luiken, Miranda Nabben, Michael Singleton, Luc Bertrand, Matthieu Ruiz, Christine Des Rosiers, Jean-Luc Balligand","doi":"10.1101/2023.11.10.564735","DOIUrl":"https://doi.org/10.1101/2023.11.10.564735","url":null,"abstract":"Background: Cardiac beta3-adrenergic receptors (beta3AR) are upregulated in diseased hearts and mediate antithetic effects to those of beta1AR and beta2AR. Beta3AR agonists were recently shown to protect from myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. The underlying mechanisms, however, remain elusive. Methods: To dissect functional, transcriptional and metabolic effects, hearts and isolated ventricular myocytes from mice harboring a moderate, cardiac-specific expression of a human ADRB3 transgene (beta3AR-Tg) and subjected to transverse aortic constriction (TAC) were assessed using echocardiography, RNAseq, PET scan, metabolomics, seahorse and metabolic flux analysis. Subsequently, signaling and metabolic pathways were investigated further in vivo in beta3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express beta3AR and subjected to neurohormonal stress. These results were completed with an analysis of single nucleus RNAseq data from human cardiac myocytes from heart failure patients. Results: Compared with WT littermate, beta3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. Beta3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the Pentose-Phosphate Pathway (PPP) in beta3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the PPP, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, while downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in beta3AR-Tg post-TAC compared to WT, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as upstream transcription factor which was functionally verified in beta3AR- expressing cardiac myocytes where its translocation and nuclear activity was dependent on beta3AR activation of nitric-oxide synthase (NOS) NO production. Conclusion: Moderate expression of cardiac beta3AR, at levels observed in human cardiac myocardium, exerts antioxidant effects through activation of the PPP and NRF2 pathway, thereby preserving myocardial oxidative metabolism, function and integrity under pathophysiological stress.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"32 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134957568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566668
Kelsey L. Anbuhl, Marielisa Diez Castro, Nikki A. Lee, Vivian S. Lee, Dan H. Sanes
We often exert greater cognitive resources (i.e., listening effort) to understand speech under challenging acoustic conditions. This mechanism can be overwhelmed in those with hearing loss, resulting in cognitive fatigue in adults, and potentially impeding language acquisition in children. However, the neural mechanisms that support listening effort are uncertain. Evidence from human studies suggest that the cingulate cortex is engaged under difficult listening conditions, and may exert top-down modulation of the auditory cortex (AC). Here, we asked whether the gerbil cingulate cortex (Cg) sends anatomical projections to the AC that facilitate perceptual performance. To model challenging listening conditions, we used a sound discrimination task in which stimulus parameters were presented in either Easy or Hard blocks (i.e., long or short stimulus duration, respectively). Gerbils achieved statistically identical psychometric performance in Easy and Hard blocks. Anatomical tracing experiments revealed a strong, descending projection from layer 2/3 of the Cg1 subregion of the cingulate cortex to superficial and deep layers of primary and dorsal AC. To determine whether Cg improves task performance under challenging conditions, we bilaterally infused muscimol to inactivate Cg1, and found that psychometric thresholds were degraded for only Hard blocks. To test whether the Cg-to-AC projection facilitates task performance, we chemogenetically inactivated these inputs and found that performance was only degraded during Hard blocks. Taken together, the results reveal a descending cortical pathway that facilitates perceptual performance during challenging listening conditions.
{"title":"Cingulate cortex facilitates auditory perception under challenging listening conditions","authors":"Kelsey L. Anbuhl, Marielisa Diez Castro, Nikki A. Lee, Vivian S. Lee, Dan H. Sanes","doi":"10.1101/2023.11.10.566668","DOIUrl":"https://doi.org/10.1101/2023.11.10.566668","url":null,"abstract":"We often exert greater cognitive resources (i.e., listening effort) to understand speech under challenging acoustic conditions. This mechanism can be overwhelmed in those with hearing loss, resulting in cognitive fatigue in adults, and potentially impeding language acquisition in children. However, the neural mechanisms that support listening effort are uncertain. Evidence from human studies suggest that the cingulate cortex is engaged under difficult listening conditions, and may exert top-down modulation of the auditory cortex (AC). Here, we asked whether the gerbil cingulate cortex (Cg) sends anatomical projections to the AC that facilitate perceptual performance. To model challenging listening conditions, we used a sound discrimination task in which stimulus parameters were presented in either Easy or Hard blocks (i.e., long or short stimulus duration, respectively). Gerbils achieved statistically identical psychometric performance in Easy and Hard blocks. Anatomical tracing experiments revealed a strong, descending projection from layer 2/3 of the Cg1 subregion of the cingulate cortex to superficial and deep layers of primary and dorsal AC. To determine whether Cg improves task performance under challenging conditions, we bilaterally infused muscimol to inactivate Cg1, and found that psychometric thresholds were degraded for only Hard blocks. To test whether the Cg-to-AC projection facilitates task performance, we chemogenetically inactivated these inputs and found that performance was only degraded during Hard blocks. Taken together, the results reveal a descending cortical pathway that facilitates perceptual performance during challenging listening conditions.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"38 21","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134953449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566664
Natalia Hernandes, Mollyann Xiaomeng Qi, Soumitra Bhide, Courtney Brown, Benjamin J Camm, Simon W Baxter, Charles Robin
BACKGROUND: One of the proposed applications of gene drives has been to revert pesticide resistant mutations back to the ancestral susceptible state. Insecticides that have become ineffective because of the rise of resistance could have reinvigorated utility and be used to suppress pest populations again, perhaps at lower application doses. RESULTS: We have created a laboratory model for susceptibility gene drives that replaces field-selected resistant variants of the acetylcholine esterase (Ace) locus of Drosophila melanogaster with ancestral susceptible variants. We constructed a CRISPR/Cas9 homing drive and found that homing occurred in many genetic backgrounds with varying efficiencies. While the drive itself could not be homozygosed, it converted resistant alleles into susceptible ones and produced recessive lethal alleles that could suppress populations. Our studies provided evidence for two distinct classes of Gene Drive Resistance (GDR): rather than being mediated by the conventional Non-Homologous End-joining (NHEJ) pathway, one seemed to involve short homologous repair and the other was defined by genetic background. Additionally, we used simulations to explore a distinct application of susceptibility drives; the use of chemicals to prevent the spread of synthetic gene drives into protected areas. CONCLUSIONS: Insecticide susceptibility gene drives could be useful tools to control pest insects however problems associated with particularities of the target loci and GDR will need to be overcome for them to be effective. Furthermore, realistic patterns of pest dispersal and high insecticide exposure rates would be required if susceptibility were to be useful as safety-switch to prevent the unwanted spread of gene drives.
{"title":"Acetylcholine esterase of Drosophila melanogaster: a laboratory model to explore applications of insecticide susceptibility gene drives","authors":"Natalia Hernandes, Mollyann Xiaomeng Qi, Soumitra Bhide, Courtney Brown, Benjamin J Camm, Simon W Baxter, Charles Robin","doi":"10.1101/2023.11.10.566664","DOIUrl":"https://doi.org/10.1101/2023.11.10.566664","url":null,"abstract":"BACKGROUND: One of the proposed applications of gene drives has been to revert pesticide resistant mutations back to the ancestral susceptible state. Insecticides that have become ineffective because of the rise of resistance could have reinvigorated utility and be used to suppress pest populations again, perhaps at lower application doses. RESULTS: We have created a laboratory model for susceptibility gene drives that replaces field-selected resistant variants of the acetylcholine esterase (Ace) locus of Drosophila melanogaster with ancestral susceptible variants. We constructed a CRISPR/Cas9 homing drive and found that homing occurred in many genetic backgrounds with varying efficiencies. While the drive itself could not be homozygosed, it converted resistant alleles into susceptible ones and produced recessive lethal alleles that could suppress populations. Our studies provided evidence for two distinct classes of Gene Drive Resistance (GDR): rather than being mediated by the conventional Non-Homologous End-joining (NHEJ) pathway, one seemed to involve short homologous repair and the other was defined by genetic background. Additionally, we used simulations to explore a distinct application of susceptibility drives; the use of chemicals to prevent the spread of synthetic gene drives into protected areas. CONCLUSIONS: Insecticide susceptibility gene drives could be useful tools to control pest insects however problems associated with particularities of the target loci and GDR will need to be overcome for them to be effective. Furthermore, realistic patterns of pest dispersal and high insecticide exposure rates would be required if susceptibility were to be useful as safety-switch to prevent the unwanted spread of gene drives.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"49 10","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566917
Magdalena Abrozek-Latecka, Piotr Kozlowski, Grazyna Hoser, Magdalena Bandyszewska, Karolina Hanusek, Dominika Nowis, Jakub Golab, Malgorzata Grzanka, Agnieszka Piekielko-Witkowska, Luise Schulz, Franziska Hornung, Stefanie Deinhardt-Emmer, Ewa Kozlowska, Tomasz Skirecki
Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger cell death and IL-1α release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1α and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1α as a potential novel component driving the pulmonary immunothromobosis in COVID-19.
{"title":"SARS-CoV-2 and its ORF3a, E and M viroporins activate inflammasome in human macrophages and induce of IL-1α in pulmonary epithelial and endothelial cells","authors":"Magdalena Abrozek-Latecka, Piotr Kozlowski, Grazyna Hoser, Magdalena Bandyszewska, Karolina Hanusek, Dominika Nowis, Jakub Golab, Malgorzata Grzanka, Agnieszka Piekielko-Witkowska, Luise Schulz, Franziska Hornung, Stefanie Deinhardt-Emmer, Ewa Kozlowska, Tomasz Skirecki","doi":"10.1101/2023.11.13.566917","DOIUrl":"https://doi.org/10.1101/2023.11.13.566917","url":null,"abstract":"Inflammasome assembly is a potent mechanism responsible for the host protection against pathogens, including viruses. When compromised, it can allow viral replication, while when disrupted, it can perpetuate pathological responses by IL-1 signaling and pyroptotic cell death. SARS-CoV-2 infection was shown to activate inflammasome in the lungs of COVID-19 patients, however, potential mechanisms responsible for this response are not fully elucidated. In this study, we investigated the effects of ORF3a, E and M SARS-CoV-2 viroporins in the inflammasome activation in major populations of alveolar sentinel cells: macrophages, epithelial and endothelial cells. We demonstrated that each viroporin is capable of activation of the inflammasome in macrophages to trigger cell death and IL-1α release from epithelial and endothelial cells. Small molecule NLRP3 inflammasome inhibitors reduced IL-1 release but weakly affected the pyroptosis. Importantly, we discovered that while SARS-CoV-2 could not infect the pulmonary microvascular endothelial cells it induced IL-1α and IL-33 release. Together, these findings highlight the essential role of macrophages as the major inflammasome-activating cell population in the lungs and point to endothelial cell expressed IL-1α as a potential novel component driving the pulmonary immunothromobosis in COVID-19.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"49 3","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Unfolded Protein Response (UPR) is a retrograde, ER-to-nucleus, signalling pathway which is conserved across kingdoms. In plants, it contributes to development, reproduction, immunity and tolerance to abiotic stress. This RNA sequencing dataset was produced from 14-day-old Arabidopsis thaliana seedlings challenged by tunicamycin (Tm), an antibiotic inhibiting Asn-linked glycosylation in the endoplasmic reticulum (ER), causing an ER stress and eventually activating the UPR. Wild-type (WT) and a double mutant deficient for two main actors of the UPR (INOSITOL-REQUIRING ENZYME 1A and INOSITOL-REQUIRING ENZYME 1B) were used as genetic backgrounds in our experimental setup, allowing to distinguish among differentially-expressed genes (DEGs) which ones are dependent on or independent on IRE1s. Also, shoots and roots were harvested separately to determine organ-specific transcriptomic responses to Tm. Library and sequencing were performed using DNBseq technology by the Beijing Genomics Institute. Reads were mapped and quantified against the Arabidopsis genome. Differentially-expressed genes were identified using Rflomics upon filtering and normalization by the Trimmed Mean of M-value (TMM) method. While the genotype effect was weak under mock conditions (with a total of 182 DEGs in shoots and 195 DEGs in roots), the tunicamycin effect on each genotype was characterized by several hundred of DEGs in both shoots and roots. Among these genes, 872 and 563 genes were statistically up- and down-regulated in the shoot tissues of the double mutant when compared to those of WT, respectively. In roots of Tm-challenged seedlings, 425 and 439 genes were significantly up- and down-regulated in mutants with respect to WT. We believe that our dataset could be reused for investigating any biological questions linked to ER homeostasis and its role in plant physiology.
{"title":"A transcriptomic dataset for investigating the Arabidopsis Unfolded Protein Response under chronic, proteotoxic endoplasmic reticulum stress","authors":"Amelie Ducloy, Marianne Azzopardi, Caroline Ivsic, Gwendal Cueff, Delphine Sourdeval, Delphine Charif, Jean-Luc Cacas","doi":"10.1101/2023.11.12.566746","DOIUrl":"https://doi.org/10.1101/2023.11.12.566746","url":null,"abstract":"The Unfolded Protein Response (UPR) is a retrograde, ER-to-nucleus, signalling pathway which is conserved across kingdoms. In plants, it contributes to development, reproduction, immunity and tolerance to abiotic stress. This RNA sequencing dataset was produced from 14-day-old Arabidopsis thaliana seedlings challenged by tunicamycin (Tm), an antibiotic inhibiting Asn-linked glycosylation in the endoplasmic reticulum (ER), causing an ER stress and eventually activating the UPR. Wild-type (WT) and a double mutant deficient for two main actors of the UPR (INOSITOL-REQUIRING ENZYME 1A and INOSITOL-REQUIRING ENZYME 1B) were used as genetic backgrounds in our experimental setup, allowing to distinguish among differentially-expressed genes (DEGs) which ones are dependent on or independent on IRE1s. Also, shoots and roots were harvested separately to determine organ-specific transcriptomic responses to Tm. Library and sequencing were performed using DNBseq technology by the Beijing Genomics Institute. Reads were mapped and quantified against the Arabidopsis genome. Differentially-expressed genes were identified using Rflomics upon filtering and normalization by the Trimmed Mean of M-value (TMM) method. While the genotype effect was weak under mock conditions (with a total of 182 DEGs in shoots and 195 DEGs in roots), the tunicamycin effect on each genotype was characterized by several hundred of DEGs in both shoots and roots. Among these genes, 872 and 563 genes were statistically up- and down-regulated in the shoot tissues of the double mutant when compared to those of WT, respectively. In roots of Tm-challenged seedlings, 425 and 439 genes were significantly up- and down-regulated in mutants with respect to WT. We believe that our dataset could be reused for investigating any biological questions linked to ER homeostasis and its role in plant physiology.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"45 21","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991747","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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